Abstract

In Utetheisa ornatrix (Lepidoptera, Arctiidae), the
female mates preferentially with larger males. Having a larger father
results in the eggs being more richly endowed with defensive
pyrrolizidine alkaloid (which the female receives from the male with
the sperm package, in quantity proportional to the male's body mass,
and passes on to the eggs); having a larger father also results in the
sons and daughters themselves being larger (body mass is heritable in
Utetheisa). We provide evidence herein that these
consequences enhance the fitness of the offspring. Eggs sired by larger
males are less vulnerable to predation (presumably because of their
higher alkaloid content), whereas sons and daughters, by virtue of
being larger, are, respectively, more successful in courtship and more
fecund. The female Utetheisa, therefore, by being
choosy, reaps both direct phenotypic and indirect genetic benefits.

In the moth Utetheisa ornatrix
(henceforth called Utetheisa), the female exercises mate
choice. She mates preferentially with large males, thereby potentially
deriving both direct phenotypic benefits and indirect genetic benefits.

The details of this reproductive strategy are complex (1).
Utetheisa, as a larva, feeds on plants of the genus
Crotalaria (family Fabaceae), containing poisonous
pyrrolizidine alkaloids (henceforth called alkaloids).
Utetheisa is insensitive to the alkaloids, and the larva
stores the chemicals systemically, retaining them through metamorphosis
into the adult stage. At mating, the male transfers a substantial
fraction of his alkaloidal load to the female with the sperm package
(spermatophore; ref. 2). The gift is transmitted by the female in part
to the eggs, together with a supplement of her own alkaloidal supply
(3). All developmental stages of Utetheisa are protected by
the alkaloid. The larvae and adults are rejected by spiders (4, 5), and
the eggs are avoided by ants (6) and coccinellid beetles (3). The
spermatophore in Utetheisa is of substantial size, amounting
on average to over 10% of male body mass (7). It also contains
nutrient, which the female invests in egg production. Females mate on
average with four to five males (8) over their lifespan of 3 to 4
weeks. With each mating, the female is able to increase her fecundity
by 15% (9). Fecundity in Utetheisa is also a function of
intrinsic female body mass: large females lay greater numbers of eggs
(9).

Female Utetheisa do not mate randomly with males but do so
selectively with males of higher alkaloid content. The female does not
gauge male alkaloid content directly but does so indirectly, on the
basis of a pheromone (hydroxydanaidal) that the male produces from
alkaloid, in proportion to his alkaloid load, and airs during
close-range precopulatory interaction with the female (2, 10). Males
richest in alkaloid, having the strongest pheromonal scent, are also
largest, and apt to bestow the largest alkaloidal (and presumably
nutritive) gifts. In essence, by selecting males of high alkaloid
content, the female is selecting males of large size.

We recently established that body mass is heritable in
Utetheisa (11). This finding indicated that by favoring
larger males, females obtain not only larger nuptial gifts but also
larger sons and daughters. The offspring, as a consequence, could
receive direct phenotypic benefits (from the nuptial gifts) and
indirect genetic benefits (from the expression of largeness in sons and
daughters). We postulated that these benefits should be measurable and
found that the offspring of preferred males do indeed fare better than
the offspring of nonpreferred males. Specifically, we showed that
(i) eggs sired by preferred males are less vulnerable to
predation; (ii) sons of preferred males are more successful
in courtship; and (iii) daughters of preferred males are
more fecund.

Our basic protocol was as follows: (i) we confined a female
with two males (one large, one small) until she chose to mate with one
of them (preferred male, primary mating); (ii) we confined a
second female with the nonpreferred male and allowed mating to take
place (secondary mating); (iii) we allowed the first and
second females to lay eggs and checked these for relative vulnerability
to predation by a coccinellid beetle (experiment 1); or alternatively,
(iv) we allowed the eggs of the first and second female to
develop into adult sons and daughters and tested the sons for relative
success in courtship trials (experiment 2) and the daughters for
relative fecundity (experiment 3).

Materials and Methods

Utetheisa.

All Utetheisa were raised in the laboratory from stock
collected near Lake Placid, Highlands County, FL.

Larval Diets.

These were of two types (10): one based on pinto beans and lacking
alkaloid [(−) diet]; the other [(+) diet] also based on pinto
beans but containing a supplement of seeds of Crotalaria
spectabilis, a major food plant of Utetheisa. Utetheisa
reared on (+) diet [herein called (+) Utetheisa] contain
the principal alkaloid in C. spectabilis, monocrotaline, at
a level (0.6 mg per adult; ref. 12) comparable to that in
Utetheisa reared on C. spectabilis plants in
nature (0.7 mg per adult; ref. 13). Utetheisa raised on (−)
diet [herein called (−) Utetheisa] contain no detectable
amount of alkaloid (10).

Adult Body Mass.

This parameter is subject to unpredictable variation, because adult
Utetheisa differ as to when, after emergence, they discharge
their meconial waste. We knew from previous studies that pupal mass on
day 7 after pupation (pupal duration is 9–11 days in
Utetheisa) is a reliable correlate of adult body mass (11),
and we use this measure herein to express adult body mass. In the
current study, adults that are said to be “size-matched” differed
by less than 5 mg in pupal mass, whereas those said to be
“different-sized” differed by at least 20 mg (or about 10%) in
pupal mass. In males, a difference of 20 mg ensured that the
individuals differed substantially in alkaloid and hydroxydanaidal
content (2, 13).

Matings.

All matings (experiments 1–3) were carried out in small,
humidified, cylindrical containers (0.35 liter). Events were
monitored visually (under red light) at intervals of at most 6
h, to check on mating success [copulation lasts 10–12 h in
Utetheisa (7)].

Oviposition.

Mated females (experiments 1–3) were individually placed in
humidified, cylindrical containers (0.35 liter), lined with wax paper,
on which they readily oviposited. For determination of lifetime
fecundity, females were allowed to oviposit in the chambers until they
died.

Larval Rearing.

For purposes of larval rearing (experiments 2 and 3), eggs from the
first 3 days of a female's output were transferred to a small
humidified chamber while still affixed to pieces of their wax paper
backing. After 7 days and after the eggs hatched, four subsets of 8–10
larvae were confined in four separate, cylindrical containers (0.1
liter) for separate parallel raising (this separation provided a
measure of control for random environmental factors exerting a
determinant effect on the larvae). Larval food supply in the chambers
was renovated every 4 days until pupation, after which, at pupal age of
7 days, the pupae were weighed.

Pupal masses provided the basis for determining average offspring body
mass. Masses were calculated separately for sons and daughters for the
offspring from each mating category (primary and secondary mating) in
experiment 2. For each set of progeny, we first calculated the mean
body mass of sons and daughters from the four larval containers and
then, from these four values, derived the son and daughter means for
that progeny. The individual progeny means, in turn, provided the basis
for calculating the overall son and daughter means for the mating
category.

Sampling of Adult Offspring.

For each set of progeny slated for fitness evaluation (experiments 2
and 3), two subsets of individuals were selected for actual assessment:
a group of three sons and three daughters randomly selected from the
sample (randomly chosen sons and daughters) and a group of three sons
and three daughters (mean-sized sons and daughters) selected to be of
average body mass (these individuals differed by no more than 5 mg from
the mean mass of their siblings). The double sampling procedure
provided a dual basis for evaluation of relative offspring fitness.

Experiment 1: Vulnerability of Eggs.

We confined 30 virgin, 3-day-old (−) Utetheisa females
individually in mating chambers with two different-sized, 3-day-old,
virgin (+) Utetheisa males. Courtship was monitored visually
for the first hour to ensure that both males made fluttering advances
to the female, as they typically do during precopulatory interaction,
when they also evert the glandular brushes bearing hydroxydanaidal
(10). When a mating took place (primary mating), the partners were
allowed to remain in copula until they spontaneously
disengaged, at which time the male was euthanized (after recording
whether he was the larger or the smaller of the pair; males were
identified by wing marks) and the female was transferred to an
oviposition chamber.

The nonpreferred male was then transferred to a second mating chamber
and paired with a size-matched sister of the first female. After mating
took place, the male was euthanized, and the female was transferred to
another oviposition chamber.

The eggs from the two females were then tested for vulnerability to
predation. We could be certain that we would be testing for the
defensive effectiveness of the father's nuptial gift, because both
mothers were (−) Utetheisa females and only the fathers
bore alkaloid.

We knew from previous work that coccinellid beetles are sensitive to
pyrrolizidine alkaloid and prone to discriminate against
Utetheisa eggs on the basis of alkaloid content (3). We
therefore chose a coccinellid beetle, Harmonia axyridis, for
our assay (14). Individual H. axyridis (prestarved for
24 h) were placed in Petri dishes (5.0-cm diameter) and offered
two egg clusters of 10 eggs each, sired respectively by the preferred
and nonpreferred males. The clusters were from the third oviposition
night of the females, and they were placed in opposite quadrants of the
dishes, still attached to pieces of their wax paper backing. At 15-min
intervals for the next 3 h, a visual count was taken of the number
of eggs of each cluster that had been eaten by the beetle. The test was
replicated 30 times (once for each set of matings) with 30 separate
beetles. We used a Wilcoxon signed rank test to compare egg loss from
the two clusters (15).

Two other values were obtained as part of this experiment, both
pertaining to the females from the primary and secondary matings:
lifetime fecundity (total egg output over the lifespan) and egg mass
(20 eggs from the third oviposition night from each female were
weighed). Comparisons were made by using paired t tests
(15).

Experiment 2: Mating Success of Sons.

A mating protocol identical to that in experiment 1 was followed,
except that the mothers and fathers were all (+) individuals. Sample
size again was 30. Offspring of the preferred and nonpreferred males
were raised separately to adulthood on (+) diet (see Larval
Rearing, above), whereupon two subsets of sons from each mating
(see Sampling of Adult Offspring, above) were put to the
test in mating assays. The assays consisted of placing one son of the
preferred male and one of the nonpreferred male together with a
3-day-old, virgin (+) female in a mating chamber and keeping a record
of which male succeeded in mating in the next 24 h (the males were
wing marked for recognition purposes).

For each trial, we determined the relative mating success of the two
types of sons, and from these scores, we calculated the overall mating
success of the sons of the two categories by using a Sign test (15).
The calculations were done separately for the randomly chosen sons and
the mean-sized sons.

Experiment 3: Fecundity of Daughters.

This experiment made use of the daughters produced in the 30 trials of
experiment 2 and involved testing for the relative fecundity of
daughters of the preferred and nonpreferred males. Mean fecundities
were first determined separately for each trial. To this end, subsets
of daughters of each category from a trial were chosen (see
Sampling of Adult Offspring, above) and assigned in pairs
(one of each category) to mate with size-matched (−) brothers.
Lifetime egg output was then tallied for each daughter, providing a
basis for calculation of the mean fecundity per category of daughter
per trial. The overall mean fecundities for the entire sample of trials
were then calculated from these means. Comparison of the overall means
for the two sets of daughters was done with a paired t test
(15). The calculations were carried out separately for the randomly
chosen daughters and the mean-sized daughters.

Body Size of Offspring of Preferred and Nonpreferred Males.

Offspring sired by preferred males (data from experiment 2 and 3) were
of significantly higher body mass than those of nonpreferred males;
sons were larger by 7.6% and daughters by 5.7% (Table
1, first two rows).

Experiment 1: Vulnerability of Eggs.

Eggs sired by preferred males proved less vulnerable to predation. On
average, by the end of 3 h, the coccinellid had eaten 13.0% more
of the eggs sired by the nonpreferred male (Table 1, third row).

Experiment 2: Mating Success of Sons.

The sons of preferred males showed a higher incidence of acceptance in
the mating trials than did the sons of nonpreferred males. This higher
incidence was true both for the randomly chosen sons (which won out in
73% of trials) and the mean-sized sons (which won out in 77% of
trials; Table 1, fourth and fifth rows).

Experiment 3: Fecundity of Daughters.

Daughters of preferred males had a significantly higher lifetime
fecundity than the daughters of nonpreferred males. On average,
randomly chosen daughters of preferred males laid an extra 5.9% eggs,
whereas the mean-sized daughters of such males laid an extra 6.1%
(Table 1, sixth and seventh rows).

Additional Data.

Data that we obtained with the two types of mated females from
experiment 1—the females from the primary mating, which had a choice
of males, and those from the secondary mating, which had no such
choice—showed that these females did not differ with respect to
lifetime fecundity (paired t test; P = 0.80)
and mean egg mass (paired t test; P = 0.41).
Moreover, from mother–offspring data from experiment 2, we established
that there is no difference for the two types of females in the
maternal heritability of body mass (calculated as previously described;
ref. 11; analysis of covariance: mother–son, P = 0.96;
mother–daughter, P = 0.87). The act of choosing
simultaneously between two males evidently has no effect on these
female reproductive parameters.

Discussion

Some of our findings lend additional support to conclusions
derived from previous work. The males chosen by the females in the
primary matings were larger on average than the nonpreferred males.
This means that the females, under the cramped quarters of our
experimental mating chambers, exercised the same criterion of mate
choice that they are known to exercise under more natural conditions
(10).

The second point concerns the body size of the progeny. The offspring
of preferred males were larger on average than those of nonpreferred
males. This finding was only to be expected, given that the preferred
males were the larger of the two fathers, and that body mass is
heritable in Utetheisa (11).

But more important was the demonstration that the offspring of
preferred males are indeed “superior.” They are superior in the
phenotypic sense, in that, as eggs, they profit defensively from
receipt of increased quantity of paternal alkaloid; they are superior
in the genetic sense, in that, as larger sons, they are apt to be more
acceptable in courtship and, as larger daughters, likely to be more
fecund. It should be noted that these results held true irrespective of
the adult sampling procedure: our randomly chosen offspring and
mean-sized offspring fared comparably in the assessments.

We did not prove directly that it is the increased quantity of
paternal alkaloid that renders the eggs of preferred males less
vulnerable. However, we do know that larger males transmit increased
quantities of alkaloid to females at mating (2) and that females bestow
increased amounts of alkaloid on eggs if the females are alkaloid-rich
(16). One could therefore expect the eggs sired by preferred males to
be of higher alkaloid content. We attribute the greater vulnerability
of eggs sired by nonpreferred males to their being underendowed with
alkaloid.

Previous studies tell us that a greater quantity of alkaloid is not the
only phenotypic benefit that the Utetheisa female receives
by choosing a larger mate. She also obtains nutrient with the
spermatophore (9); because spermatophore size varies in accordance to
male body mass (7), large males can be expected to bestow more nutrient
at mating. By accessing larger males, therefore, the female gains extra
nutrient for potential investment in egg production.

Earlier work had also shown that the female herself profits from
receipt of the male's alkaloidal gift. She does not transmit the
entire gift to the eggs but retains some of the alkaloid systemically
for her own protection (16). It has been shown experimentally that
Utetheisa females devoid of alkaloid, and therefore
defenseless vis á vis spiders, are rendered invulnerable to such
predators from the very moment they uncouple from their
alkaloid-donating mating partner (17).

In discussions of sexual selection in animals, it is customary
not only to recognize the two primary benefits accrued by the choosing
mate—the direct phenotypic benefits and the indirect genetic
benefits—but also to distinguish between genetic benefits of two kinds
(18, 19). One type, Fisherian benefits, involves the expression in the
sons of the trait that, in the father, provided the key to success in
courtship. The other type, “good genes” benefits, also a
consequence of female mate choice, involves expression in both sons and
daughters of general improvements in quality (increased fecundity,
viability).

In Utetheisa, the genetic benefits are essentially a
combination of the two types. Size being heritable in this moth means
that by selecting appropriately in courtship, the female is able to
bestow on her sons the very quality of largeness that accounted for the
success of the father and on her daughters the largeness, which in the
female is linked to fecundity. To “top it all,” the female
receives phenotypic benefits as well. The reproductive stakes at issue
for the female Utetheisa as she appraises her suitor are
evidently multiple and high.

Acknowledgments

We are indebted to H. K. Reeve for his input during the study
and for critical comments on the manuscript. M. C. B.
Andrade, H. E. Farris, and M. Servedio provided helpful comments
on earlier versions of the paper. We also thank J. Schlesinger for
technical assistance. Research support was provided by National
Institutes of Health Grant AI02908 (to T.E.) and a predoctoral
fellowship from the National Science Foundation (to V.I.). This paper
is no. 166 in the series Defense Mechanisms of Arthropods.

Footnotes

↵* To whom reprint requests should be addressed. E-mail:
te14{at}cornell.edu.

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